FOXO3: at the crossroads of metabolic, inflammatory, and tumorigenic remodeling in the colon.

The Forkhead box O3 (FOXO3) transcription factor regulates the expression of genes critical for diverse cellular functions in homeostasis. Diminished FOXO3 activity is associated with human diseases such as obesity, metabolic diseases, inflammatory diseases, and cancer. In the mouse colon, FOXO3 deficiency leads to an inflammatory immune landscape and dysregulated molecular pathways, which, under various insults, exacerbates inflammation and tumor burden, mimicking characteristics of human diseases. This deficiency also results in dysregulated lipid metabolism, and consequently, the accumulation of intracellular lipid droplets (LDs) in colonic epithelial cells and infiltrated immune cells. FOXO3 and LDs form a self-reinforcing negative regulatory loop in colonic epithelial cells, neutrophils, and macrophages, which is associated with inflammatory bowel disease and colon cancer, particularly in the context of obesity.

FOXO3 Deficiency in Neutrophils Drives Colonic Inflammation and Tumorigenesis.

Inflammatory bowel disease (IBD), characterized by infiltration of polymorphonuclear neutrophils (PMNs), increases the risk of colon cancer. PMN activation corresponds to the accumulation of intracellular Lipid Droplets (LDs). As increased LDs are negatively regulated by transcription factor Forkhead Box O3 (FOXO3), we aim to determine the significance of this regulatory network in PMN-mediated IBD and tumorigenesis. Affected tissue of IBD and colon cancer patients, colonic and infiltrated immune cells, have increased LDs' coat protein, PLIN2. Mouse peritoneal PMNs with stimulated LDs and FOXO3 deficiency have elevated transmigratory activity. Transcriptomic analysis of these FOXO3-deficient PMNs showed differentially expressed genes (DEGs; FDR < 0.05) involved in metabolism, inflammation, and tumorigenesis. Upstream regulators of these DEGs, similar to colonic inflammation and dysplasia in mice, were linked to IBD and human colon cancer. Additionally, a transcriptional signature representing FOXO3-deficient PMNs (PMN-FOXO3389) separated transcriptomes of affected tissue in IBD (p = 0.00018) and colon cancer (p = 0.0037) from control. Increased PMN-FOXO3389 presence predicted colon cancer invasion (lymphovascular p = 0.015; vascular p = 0.046; perineural p = 0.03) and poor survival. Validated DEGs from PMN-FOXO3389 (P2RX1, MGLL, MCAM, CDKN1A, RALBP1, CCPG1, PLA2G7) are involved in metabolism, inflammation, and tumorigenesis (p < 0.05). These findings highlight the significance of LDs and FOXO3-mediated PMN functions that promote colonic pathobiology.

Reduced mitochondrial activity in colonocytes facilitates AMPKα2-dependent inflammation.

Intestinal inflammation is associated with low levels of mucosal ATP, highlighting the importance of mitochondrial function associated with ATP production in the pathophysiology of the disease. In the inflamed colon of humans and mice, we found decreased levels of mitochondrial complex cytochrome c oxidase I/IV and lower ATP levels. Thus, we generated colonic ρ0 cells with reduced mitochondrial function linked to ATP production by selective depletion of mitochondrial DNA. In these cells, RNA sequencing revealed a substantial number of differentially expressed transcripts, among which 240 belonged to inflammatory pathways activated in human inflamed colon and TNF-α-treated cells (false discovery rate < 0.05). TNF-α treatment of colonic ρ0 cells augmented IL-8 expression by 9-fold (P < 0.01) via NF-κB compared to TNF-α-treated control. Moreover, reduced mitochondrial function facilitated TNF-α-mediated NF-κB luciferase promoter activity as a result of lowered inhibitory IκBα (nuclear factor of κ light polypeptide gene enhancer in B-cell inhibitor, α), leading to elevated NF-κB. In cells with reduced mitochondrial function, TNF-α facilitated AMPKα2 activation by 8-fold (P < 0.01), which was involved in NF-κB-dependent IL-8 expression. Last, in human and mouse colon, anti-TNF-α treatment restored reduced mitochondria-dependent inflammation. We propose that selective targeting of this novel mechanism provides new treatment opportunities for intestinal inflammation.-Heller, S., Penrose, H. M., Cable, C., Biswas, D., Nakhoul, H., Baddoo, M., Flemington, E., Crawford, S. E., Savkovic, S. D. Reduced mitochondrial activity in colonocytes facilitates AMPKα2-dependent inflammation.

High-fat diet induced leptin and Wnt expression: RNA-sequencing and pathway analysis of mouse colonic tissue and tumors.

Obesity, an immense epidemic affecting approximately half a billion adults, has doubled in prevalence in the last several decades. Epidemiological data support that obesity, due to intake of a high-fat, western diet, increases the risk of colon cancer; however, the mechanisms underlying this risk remain unclear. Here, utilizing next generation RNA sequencing, we aimed to determine the high-fat diet (HFD) mediated expression profile in mouse colon and the azoxymethane/dextran sulfate sodium model of colon cancer. Mice on HFD had significantly higher colonic inflammation, tumor burden, and a number of differentially expressed transcripts compared to mice on regular diet (RD). We identified 721 transcripts differentially expressed in mouse HFD colon that were in a shared pattern with colonic tumors (RD and HFD). Importantly, in mouse colon, HFD stimulated an expression signature strikingly similar to human colon cancer, especially those with inflammatory microsatellite instability. Furthermore, pathway analysis of these transcripts demonstrated their association with active inflammation and colon cancer signaling, with leptin and Wnt as the top two transcripts elevated in mouse HFD colon shared with tumors. Moreover, in mouse colon, HFD-stimulated tumorigenic Wnt pathway activation was further validated by upregulation of ß-catenin transcriptional targets. Finally, in human colon cancer, upregulation of leptin pathway members was shown with a large network of dysregulated transcripts being linked with worse overall survival.

Intestinal inflammation requires FOXO3 and prostaglandin E2-dependent lipogenesis and elevated lipid droplets.

Intestinal inflammation has been recently characterized by the dysregulation of lipids as metabolic and energy sources, revealing a novel feature of its pathophysiology. Because intracellular lipids, stored in dynamic lipid droplets (LDs), provide energy for cellular needs, we investigated whether they play a role in intestinal inflammation. In the inflamed intestine of mice, elevated LDs were found in colonic and infiltrating immune cells as shown by staining for the LD coat protein PLIN2 and for lipids with BODIPY. In colonic cells, TNF stimulated LD increases by receptor signaling rely on phosphatidylinositol 3-kinase activation. Downstream, TNF triggered a negative regulatory loop between LDs and the transcription factor FOXO3. This was shown in the colon of Foxo3-deficient mice, where elevation in PLIN2 and lipids were further facilitated by inflammation and were more prominent relative to wild-type, whereas, in colonic cells, inhibition of lipogenesis blocked the TNF-mediated loss of FOXO3. Furthermore, blockade of PGE2 synthesis abrogated TNF-stimulated increases in LDs and FOXO3 inactivation. We found in colonic tissue of Foxo3-deficient mice higher levels of cyclooxygenase-2, a mediator of prostaglandin E2 (PGE2) synthesis, supporting involvement of PGE2 in the LD-FOXO3 regulatory loop. Ultimately, TNF-stimulated lipogenesis leading to elevated LDs facilitated NF-κB-mediated increases in IL-8 protein, which is associated with the surface of LDs found in the lumina of the endoplasmic reticulum and Golgi apparatus. This novel immunometabolic mechanism of colonic inflammation involving elevated LDs could provide opportunities for new treatment options.

Epidermal growth factor receptor mediated proliferation depends on increased lipid droplet density regulated via a negative regulatory loop with FOXO3/Sirtuin6.

The proliferation of colon cancer cells is mediated in part by epidermal growth factor receptor (EGFR) signaling and requires sustained levels of cellular energy to meet its high metabolic needs. Intracellular lipid droplets (LDs) are a source of energy used for various cellular functions and they are elevated in density in human cancer, yet their regulation and function are not well understood. Here, in human colon cancer cells, EGF stimulates increases in LD density, which depends on EGFR expression and activation as well as the individual cellular capacity for lipid synthesis. Increases in LDs are blockaded by inhibition of PI3K/mTOR and PGE2 synthesis, supporting their dependency on select upstream pathways. In colon cancer cells, silencing of the FOXO3 transcription factor leads to down regulation of SIRT6, a negative regulator of lipid synthesis, and consequent increases in the LD coat protein PLIN2, revealing that increases in LDs depend on loss of FOXO3/SIRT6. Moreover, EGF stimulates loss of FOXO3/SIRT6, which is blockaded by the inhibition of upstream pathways as well as lipid synthesis, revealing existence of a negative regulatory loop between LDs and FOXO3/SIRT6. Elevated LDs are utilized by EGF treatment and their depletion through the inhibition of lipid synthesis or silencing of PLIN2 significantly attenuates proliferation. This novel mechanism of proliferative EGFR signaling leading to elevated LD density in colon cancer cells could potentially be therapeutically targeted for the treatment of tumor progression.

FOXO3 growth inhibition of colonic cells is dependent on intraepithelial lipid droplet density.

Forkhead transcription factor FOXO3 plays a critical role in suppressing tumor growth, in part, by increasing the cell cycle inhibitor p27kip1, and Foxo3 deficiency in mice results in marked colonic epithelial proliferation. Here, we show in Foxo3-deficient colonic epithelial cells a striking increase in intracytoplasmic lipid droplets (LDs), a dynamic organelle recently observed in human tumor tissue. Although the regulation and function of LDs in non-adipocytes is unclear, we hypothesize that the anti-proliferative effect of FOXO3 was dependent on lowering LD density, thus decreasing fuel energy in both normal and colon cancer cells. In mouse colonic tumors, we found an increased expression of LD coat protein PLIN2 compared with normal colonic epithelial cells. Stimulation of LD density in human colon cancer cells led to a PI3K-dependent loss of FOXO3 and a decrease in the negative regulator of lipid metabolism in Sirtuin6 (SIRT6). Foxo3 deficiency also led to a decrease in SIRT6, revealing the existence of LD and FOXO3 feedback regulation in colonic cells. In parallel, LD-dependent loss of FOXO3 led to its dissociation from the promoter and decreased expression of the cell cycle inhibitor p27kip1. Stimulation of LD density promoted proliferation in colon cancer cells, whereas silencing PLIN2 or overexpression of FOXO3 inhibited proliferation. Taken together, FOXO3 and LDs might serve as new targets for therapeutic intervention of colon cancer.

Comprehensive Transcriptomic Analysis of EWSR1::WT1 Targets Identifies CDK4/6 Inhibitors as an Effective Therapy for Desmoplastic Small Round Cell Tumors.

Desmoplastic small round cell tumors (DSRCT) are a type of aggressive, pediatric sarcoma characterized by the EWSR1::WT1 fusion oncogene. Targeted therapies for DSRCT have not been developed, and standard multimodal therapy is insufficient, leading to a 5-year survival rate of only 15% to 25%. Here, we depleted EWSR1::WT1 in DSRCT and established its essentiality in vivo. Transcriptomic analysis revealed that EWSR1::WT1 induces unique transcriptional alterations compared with WT1 and other fusion oncoproteins and that EWSR1::WT1 binding directly mediates gene upregulation. The E-KTS isoform of EWSR1::WT1 played a dominant role in transcription, and it bound to the CCND1 promoter and stimulated DSRCT growth through the cyclin D-CDK4/6-RB axis. Treatment with the CDK4/6 inhibitor palbociclib successfully reduced growth in two DSRCT xenograft models. As palbociclib has been approved by the FDA for the treatment of breast cancer, these findings demonstrate the sensitivity of DSRCT to palbociclib and support immediate clinical investigation of palbociclib for treating this aggressive pediatric cancer. SIGNIFICANCE: EWSR1::WT1 is essential for desmoplastic small round cell tumors and upregulates the cyclin D-CDK4/6-RB axis that can be targeted with palbociclib, providing a targeted therapeutic strategy for treating this deadly tumor type.

Epiploic Adipose Tissue (EPAT) in Obese Individuals Promotes Colonic Tumorigenesis: A Novel Model for EPAT-Dependent Colorectal Cancer Progression.

The obesity epidemic is associated with increased colorectal cancer (CRC) risk and progression, the mechanisms of which remain unclear. In obese individuals, hypertrophic epiploic adipose tissue (EPAT), attached to the colon, has unique characteristics compared to other fats. We hypothesized that this understudied fat could serve as a tumor-promoting tissue and developed a novel microphysiological system (MPS) for human EPAT-dependent colorectal cancer (CRC-MPS). In CRC-MPS, obese EPAT, unlike lean EPAT, considerably attracted colon cancer HT29-GFP cells and enhanced their growth. Conditioned media (CM) from the obese CRC-MPS significantly increased the growth and migration of HT29 and HCT116 cells (p < 0.001). In HT29 cells, CM stimulated differential gene expression (hOEC867) linked to cancer, tumor morphology, and metabolism similar to those in the colon of high-fat-diet obese mice. The hOEC867 signature represented pathways found in human colon cancer. In unsupervised clustering, hOEC867 separated transcriptomes of colon cancer samples from normal with high significance (PCA, p = 9.6 × 10-11). These genes, validated in CM-treated HT29 cells (p < 0.05), regulate the cell cycle, cancer stem cells, methylation, and metastasis, and are similarly altered in human colon cancer (TCGA). These findings highlight a tumor-promoting role of EPAT in CRC facilitated with obesity and establishes a platform to explore critical mechanisms and develop effective treatments.

Unraveling the Complex Interconnection between Specific Inflammatory Signaling Pathways and Mechanisms Involved in HIV-Associated Colorectal Oncogenesis.

The advancement of HIV treatment has led to increased life expectancy. However, people living with HIV (PLWH) are at a higher risk of developing colorectal cancers. Chronic inflammation has a key role in oncogenesis, affecting the initiation, promotion, transformation, and advancement of the disease. PLWH are prone to opportunistic infections that trigger inflammation. It has been documented that 15-20% of cancers are triggered by infections, and this percentage is expected to be increased in HIV co-infections. The incidence of parasitic infections such as helminths, with Ascariasis being the most common, is higher in HIV-infected individuals. Cancer cells and opportunistic infections drive a cascade of inflammatory responses which assist in evading immune surveillance, making them survive longer in the affected individuals. Their survival leads to a chronic inflammatory state which further increases the probability of oncogenesis. This review discusses the key inflammatory signaling pathways involved in disease pathogenesis in HIV-positive patients with colorectal cancers. The possibility of the involvement of co-infections in the advancement of the disease, along with highlights on signaling mechanisms that can potentially be utilized as therapeutic strategies to prevent oncogenesis or halt cancer progression, are addressed.

FOXO3 Expression in Macrophages Is Lowered by a High-Fat Diet and Regulates Colonic Inflammation and Tumorigenesis.

Obesity, characterized by augmented inflammation and tumorigenesis, is linked to genetic predispositions, such as FOXO3 polymorphisms. As obesity is associated with aberrant macrophages infiltrating different tissues, including the colon, we aimed to identify FOXO3-dependent transcriptomic changes in macrophages that drive obesity-mediated colonic inflammation and tumorigenesis. We found that in mouse colon, high-fat-diet-(HFD)-related obesity led to diminished FOXO3 levels and increased macrophages. Transcriptomic analysis of mouse peritoneal FOXO3-deficient macrophages showed significant differentially expressed genes (DEGs; FDR < 0.05) similar to HFD obese colons. These DEG-related pathways, linked to mouse colonic inflammation and tumorigenesis, were similar to those in inflammatory bowel disease (IBD) and human colon cancer. Additionally, we identified a specific transcriptional signature for the macrophage-FOXO3 axis (MAC-FOXO382), which separated the transcriptome of affected tissue from control in both IBD (p = 5.2 × 10−8 and colon cancer (p = 1.9 × 10−11), revealing its significance in human colonic pathobiologies. Further, we identified (heatmap) and validated (qPCR) DEGs specific to FOXO3-deficient macrophages with established roles both in IBD and colon cancer (IL-1B, CXCR2, S100A8, S100A9, and TREM1) and those with unexamined roles in these colonic pathobiologies (STRA6, SERPINH1, LAMB1, NFE2L3, OLR1, DNAJC28 and VSIG10). These findings establish an important understanding of how HFD obesity and related metabolites promote colonic pathobiologies.

Tumor suppressor FOXO3 mediates signals from the EGF receptor to regulate proliferation of colonic cells.

Epithelial proliferation, critical for homeostasis, healing, and colon cancer progression, is in part controlled by epidermal growth factor receptor (EGFR). Proliferation of colonic epithelia can be induced by Citrobacter rodentium infection, and we have demonstrated that activity of tumor suppressor FOXO3 was attenuated after this infection. Thus the aim of this study was to determine the contribution of FOXO3 in EGFR-dependent proliferation of intestinal epithelia and colon cancer cell lines. In this study we show that, during infection with C. rodentium, EGFR was significantly phosphorylated in colonic mucosa and Foxo3 deficiency in this model lead to an increased number of bromodeoxyuridine-positive cells. In vitro, in human colon cancer cells, increased expression and activation of EGFR was associated with proliferation that leads to FOXO3 phosphorylation (inactivation). Following EGFR activation, FOXO3 was phosphorylated (via phosphatidylinositol 3-kinase/Akt) and translocated to the cytosol where it was degraded. Moreover, inhibition of proliferation by overexpressing FOXO3 was not reversed by the EGFR signaling, implicating FOXO3 as one of the regulators downstream of EGFR. FOXO3 binding to the promoter of the cell cycle inhibitor p27kip1 was decreased by EGFR signaling, suggesting its role in EGFR-dependent proliferation. In conclusion, we show that proliferation in colonic epithelia and colon cancer cells, stimulated by EGFR, is mediated via loss of FOXO3 activity and speculate that FOXO3 may serve as a target in the development of new pharmacological treatments of proliferative diseases.

Genistein inhibits proliferation of colon cancer cells by attenuating a negative effect of epidermal growth factor on tumor suppressor FOXO3 activity.

BACKGROUND: Soy consumption is associated with a lower incidence of colon cancer which is believed to be mediated by one of its of components, genistein. Genistein may inhibit cancer progression by inducing apoptosis or inhibiting proliferation, but mechanisms are not well understood. Epidermal growth factor (EGF)-induced proliferation of colon cancer cells plays an important role in colon cancer progression and is mediated by loss of tumor suppressor FOXO3 activity. The aim of this study was to assess if genistein exerts anti-proliferative properties by attenuating the negative effect of EGF on FOXO3 activity. METHODS: The effect of genistein on proliferation stimulated by EGF-mediated loss of FOXO3 was examined in human colonic cancer HT-29 cells. EGF-induced FOXO3 phosphorylation and translocation were assessed in the presence of genistein. EGF-mediated loss of FOXO3 interactions with p53 (co-immunoprecipitation) and promoter of p27kip1 (ChIP assay) were examined in presence of genistein in cells with mutated p53 (HT-29) and wild type p53 (HCT116). Silencing of p53 determined activity of FOXO3 when it is bound to p53. RESULTS: Genistein inhibited EGF-induced proliferation, while favoring dephosphorylation and nuclear retention of FOXO3 (active state) in colon cancer cells. Upstream of FOXO3, genistein acts via the PI3K/Akt pathway to inhibit EGF-stimulated FOXO3 phosphorylation (i.e. favors active state). Downstream, EGF-induced disassociation of FOXO3 from mutated tumor suppressor p53, but not wild type p53, is inhibited by genistein favoring FOXO3-p53(mut) interactions with the promoter of the cell cycle inhibitor p27kip1 in colon cancer cells. Thus, the FOXO3-p53(mut) complex leads to elevated p27kip1 expression and promotes cell cycle arrest. CONCLUSION: These novel anti-proliferative mechanisms of genistein suggest a possible role of combining genistein with other chemoreceptive agents for the treatment of colon cancer.

Elevated ATGL in colon cancer cells and cancer stem cells promotes metabolic and tumorigenic reprogramming reinforced by obesity.

Obesity is a worldwide epidemic associated with increased risk and progression of colon cancer. Here, we aimed to determine the role of adipose triglyceride lipase (ATGL), responsible for intracellular lipid droplet (LD) utilization, in obesity-driven colonic tumorigenesis. In local colon cancer patients, significantly increased ATGL levels in tumor tissue, compared to controls, were augmented in obese individuals. Elevated ATGL levels in human colon cancer cells (CCC) relative to non-transformed were augmented by an obesity mediator, oleic acid (OA). In CCC and colonospheres, enriched in colon cancer stem cells (CCSC), inhibition of ATGL prevented LDs utilization and inhibited OA-stimulated growth through retinoblastoma-mediated cell cycle arrest. Further, transcriptomic analysis of CCC, with inhibited ATGL, revealed targeted pathways driving tumorigenesis, and high-fat-diet obesity facilitated tumorigenic pathways. Inhibition of ATGL in colonospheres revealed targeted pathways in human colonic tumor crypt base cells (enriched in CCSC) derived from colon cancer patients. In CCC and colonospheres, we validated selected transcripts targeted by ATGL inhibition, some with emerging roles in colonic tumorigeneses (ATG2B, PCK2, PGAM1, SPTLC2, IGFBP1, and ABCC3) and others with established roles (MYC and MUC2). These findings demonstrate obesity-promoted, ATGL-mediated colonic tumorigenesis and establish the therapeutic significance of ATGL in obesity-reinforced colon cancer progression.

Ulcerative colitis immune cell landscapes and differentially expressed gene signatures determine novel regulators and predict clinical response to biologic therapy.

The heterogeneous pathobiology underlying Ulcerative Colitis (UC) is not fully understood. Using publicly available transcriptomes from adult UC patients, we identified the immune cell landscape, molecular pathways, and differentially expressed genes (DEGs) across patient cohorts and their association with treatment outcomes. The global immune cell landscape of UC tissue included increased neutrophils, T CD4 memory activated cells, active dendritic cells (DC), and M0 macrophages, as well as reduced trends in T CD8, Tregs, B memory, resting DC, and M2 macrophages. Pathway analysis of DEGs across UC cohorts demonstrated activated bacterial, inflammatory, growth, and cellular signaling. We identified a specific transcriptional signature of one hundred DEGs (UC100) that distinctly separated UC inflamed from uninflamed transcriptomes. Several UC100 DEGs, with unidentified roles in UC, were validated in primary tissue. Additionally, non-responders to anti-TNFα and anti-α4ß7 therapy displayed distinct profiles of immune cells and pathways pertaining to inflammation, growth, and metabolism. We identified twenty resistant DEGs in UC non-responders to both therapies of which four had significant predictive power to treatment outcome. We demonstrated the global immune landscape and pathways in UC tissue, highlighting a unique UC signature across cohorts and a UC resistant signature with predictive performance to biologic therapy outcome.

Bacterial TLR4 and NOD2 signaling linked to reduced mitochondrial energy function in active inflammatory bowel disease.

Inflammatory bowel disease (IBD) has been linked to active signaling with bacterial components and reduced mitochondrial ATP production; however, synergism between both of these disease characteristics remains unclear. We aimed to determine in human IBD transcriptomes the link between a transcriptional signature unique to intestinal cells (ICs) with reduced mitochondrial ATP production (Mito-0) and bacteria triggered signaling using a bioinformatics approach. We generated an IC Mito-0 panel comprised of 199 differentially expressed (DE) transcripts mediated by reduced mitochondrial ATP function (DEGseq, log2 fold-change > |2|, p < .001). Transcripts from this panel were involved in diverse biological functions including regulation of mitochondrial energy (lower ATP), extracellular matrix, cell-cell contact, cytoskeleton, growth, metabolism, and inflammation. Next, unsupervised hierarchical clustering showed that the Mito-0 panel distinctly separated inflamed IBD from non-inflamed transcriptomes, which was also supported by principal component analysis (PCA) revealing distinct variation between sample types based on presence of the Mito-0 signature (PCA, p = 8.77e-09). Utilizing three independent IBD cohorts, we validated that 60 novel transcripts from the Mito-0 panel were significantly increased in inflamed tissue. Subsequently, KEGG generated bacterial TLR4 and NOD2 transcriptional signatures strongly associated with inflamed IBD transcriptomes and with the Mito-0 signature as determined by Spearman's analysis (coefficient of correlation, r = 0.92, p < .05). Herein, using a comprehensive analysis we demonstrated existence of an axis between bacteria triggered signaling and reduced mitochondrial energy function. Furthermore, we identified and validated novel transcripts within this axis as potential drivers and therapeutic targets for human IBD.

Tumor suppressor FOXO3 participates in the regulation of intestinal inflammation.

Inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, is characterized by chronic mucosal injury and the infiltration of inflammatory cells. Tumor suppressor FOXO3 regulates gene expression and its translocation to the cytosol leads to the abrogation of its transcriptional function. We have previously shown that bacterial infection regulates FOXO3 in intestinal epithelial cells and increases cytokine levels. As TNFalpha is a major contributor in intestinal inflammation, the aim of this study was to assess its effect on FOXO3 and FOXO3's contribution to intestinal inflammation in vitro and in vivo. TNFalpha induces the translocation of nuclear FOXO3 into the cytosol where it undergoes proteasomal degradation in human intestinal HT-29 cells. Proximally, the PI3K and IKK pathways mediate TNFalpha-induced FOXO3 phosphorylation. In FOXO3-silenced HT-29 cells, TNFalpha-induced IL-8 expression is increased approximately 83%. In vivo, Foxo3 is present in the nuclei and cytosol of colonic crypt epithelia. In DSS-induced colonic inflammation, Foxo3's nuclear localization is lost and it is only found in the cytosol. Consistent with a role for Foxo3 in colitis, Foxo3-deficient mice treated with DSS developed more severe colonic inflammation with an increased number of intraepithelial lymphocytes and PMNs infiltrated in the epithelia, than wild-type mice. In summary, TNFalpha inactivates FOXO3 in intestinal epithelia through the PI3K and IKK pathways and FOXO3 inactivation leads to the upregulation of IL-8 in vitro; in vivo Foxo3 is in the cytosol of inflamed colonic epithelia and Foxo3 deficiency leads to severe intestinal inflammation.

Loss of Forkhead Box O3 Facilitates Inflammatory Colon Cancer: Transcriptome Profiling of the Immune Landscape and Novel Targets.

BACKGROUND & AIMS: Diminished forkhead box O3 (FOXO3) function drives inflammation and cancer growth; however, mechanisms fostering these pathobiologies are unclear. Here, we aimed to identify in colon loss of FOXO3-dependent cellular and molecular changes that facilitate inflammation-mediated tumor growth. METHODS: FOXO3 knockout (KO) and wild-type (WT) mice were used in the AOM/DSS model of inflammation-mediated colon cancer. Bioinformatics were used for profiling of mRNA sequencing data from human and mouse colon and tumors; specific targets were validated in human colon cancer cells (shFOXO3). RESULTS: In mice, FOXO3 deficiency led to significantly elevated colonic tumor burden (incidence and size) compared with WT (P < .05). In FOXO3 KO colon, activated molecular pathways overlapped with those associated with mouse and human colonic inflammation and cancer, especially human colonic tumors with inflammatory microsatellite instability (false discovery rate < 0.05). FOXO3 KO colon, similar to tumors, had increased neutrophils, macrophages, B cells, T cells, and decreased natural killer cells (false discovery rate < 0.05). Moreover, in KO colon differentially expressed transcripts were linked to activation of inflammatory nuclear factor kappa B, tumorigenic cMyc, and bacterial Toll-like receptor signaling. Among differentially expressed transcripts, we validated altered expression of integrin subunit alpha 2 (ITGA2), ADAM metallopeptidase with thrombospondin type 1 motif 12, and ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 5 in mouse WT and FOXO3 KO colon and tumors (P < .05). Similarly, their altered expression was found in human inflammatory bowel disease and colon cancer tissues and linked to poor patient survival. Ultimately, in human colon cancer cells, FOXO3 knockdown (shFOXO3) led to significantly increased ITGA2, and silencing ITGA2 (siRNA) alone diminished cell growth. CONCLUSIONS: We identified the loss of FOXO3-mediated immune landscape, pathways, and transcripts that could serve as biomarkers and new targets for inflammatory colon cancer treatment.

Human Adipose-Derived Hydrogel Characterization Based on In Vitro ASC Biocompatibility and Differentiation.

Hydrogels serve as three-dimensional scaffolds whose composition can be customized to allow attachment and proliferation of several different cell types. Extracellular matrix-derived hydrogels are considered close replicates of the tissue microenvironment. They can serve as scaffolds for in vitro tissue engineering and are a useful tool to study cell-scaffold interaction. The aim of the present study was to analyze the effect of adipose-derived stromal/stem cells (ASCs) and decellularized adipose tissue-derived (DAT) hydrogel interaction on ASC morphology, proliferation, differentiation, and DAT hydrogel microstructure. First, the ASCs were characterized using flow cytometry, adipogenic/osteogenic differentiation, colony-forming unit fibroblast assay and doubling time. The viability and proliferation assays showed that ASCs seeded in DAT hydrogel at different concentrations and cultured for 21 days remained viable and displayed proliferation. ASCs were seeded on DAT hydrogel and cultured in stromal, adipogenic, or osteogenic media for 14 or 28 days. The analysis of adipogenic differentiation demonstrated the upregulation of adipogenic marker genes and accumulation of oil droplets in the cells. Osteogenic differentiation demonstrated the upregulation of osteogenic marker genes and mineral deposition in the DAT hydrogel. The analysis of DAT hydrogel fiber metrics revealed that ASC seeding, and differentiation altered both the diameter and arrangement of fibers in the matrix. Matrix metalloproteinase-2 (MMP-2) activity was assessed to determine the possible mechanism for DAT hydrogel remodeling. MMP-2 activity was observed in all ASC seeded samples, with the osteogenic samples displaying the highest MMP-2 activity. These findings indicate that DAT hydrogel is a cytocompatible scaffold that supports the adipogenic and osteogenic differentiation of ASCs. Furthermore, the attachment of ASCs and differentiation along adipogenic and osteogenic lineages remodels the microstructure of DAT hydrogel.

Tumor suppressor Foxo3a is involved in the regulation of lipopolysaccharide-induced interleukin-8 in intestinal HT-29 cells.

Enteric bacteria and their products play an important role in intestinal inflammation; however, the complete mechanisms are not elucidated yet. Tumor suppressor Foxo3a regulates gene expression in the nucleus, and its translocation to the cytosol leads to inactivation. Proximally, Foxo3a is regulated by different pathways including the phosphoinositide 3-kinase (PI3K) pathway. The aim of this study was to determine the effect of bacterial infection on Foxo3a in intestinal epithelial cells and to examine the contribution of Foxo3a in intestinal inflammation. Bacterial lipopolysaccharide (LPS) and infection with mouse pathogen Citrobacter rodentium induce translocation of the nuclear Foxo3a into the cytosol, where it degrades in human HT-29 and mouse CMT-93 cells. In colonic epithelia of healthy mice, Foxo3a is localized in the epithelia at the bottom of the crypts in both the nucleus and the cytosol, while in C. rodentium-infected colon Foxo3a is expressed along the crypts and located mainly in the cytosol, suggesting its inactivation. LPS utilized the PI3K pathway to inhibit Foxo3a. Additionally, inhibition of PI3K attenuated LPS-induced proinflammatory interleukin-8 (IL-8). LPS-induced IL-8 is increased in HT-29 cells with silenced Foxo3a. Moreover, in HT-29 cells with silenced Foxo3a, the amount of IkappaBalpha, an NF-kappaB inhibitor, is decreased. In conclusion, LPS and bacterial infection inactivate Foxo3a in intestinal epithelia via the PI3K pathway and inactivated Foxo3a leads to the upregulation of IL-8 by suppressing inhibitory IkappaBalpha.